Isolation and purification of large quantities of DNA replication intermediates by pH step alkaline elution

Abstract

The alkaline elution technique has been modified to be used in the isolation of DNA replication intermediates and in the study of the process of DNA replication. In this procedure pulse labeled CHO cells are layered onto a membrane filter, lysed with detergent, and the nascent DNA eluted in step-wise fashion with tetrapropylammonium hydroxide at pH 11.0, 11.3, 11.5 and 12.1. Alkaline sucrose sedimentation of the eluted DNA shows that the pH 11.0 material consists of less than 9S fragments consistant with those described by Okazaki and others. DNA eluting at pH 11.3 has a molecular weight of 8-12 million daltons, DNA which elutes at pH 11.5 sediments with a molecular weight of 20-30 million daltons. Two independent lines of evidence suggest that the pH 11.3 material includes DNA sequences synthesized at replicon origins. (1) Exposure of cells to low doses of X-ray prior to pulse labeling reduces the pH 11.3 fraction by 40-50% while there is little change in the other fractions. (2) Synchronization of cells by inhibiting DNA synthesis with FdU, followed by a 2 min pulse label, yields approximately 50% of the incorporated 3H-thymidine in the pH 11.3 fraction. The pH step elution technique has the following advantages: 1. Intermediates of high specific activity can be isolated from 10(6) cells per filter; 2. By lysing cells on a filter, proteins, nucleases, and other cellular materials are eliminated; 3. DNA in the lysate is never handled, thus eliminating shearing; 4. Eluted DNA may be instantaneously neutralized by collecting into a buffer to protect it from alkaline degradation.